The ␥-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptormediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of ␥-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membranebound TNFR1 C-terminal fragment is subsequently cleaved by ␥-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient ␥-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and ␥-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a ␥-secretase substrate and suggest that ␥-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the ␥-secretase protease to pro-inflammatory cytokine signaling.The biological activities of the tumor necrosis factor-␣ (TNF) pro-inflammatory cytokine are resolved by two distinct cell surface receptors, TNFR1 3 and TNFR2, which elicit a diversity of cellular responses, such as inflammation, cell proliferation, cell differentiation, and initiation of apoptosis (1-10). TNFR1 initiates either pro-inflammatory or pro-apoptotic signaling through the selective recruitment of intracellular adaptor and effector proteins (1,3,6,7,11). Ligand binding and trimerization of TNFR1 enables the recruitment of TNFR1-associated death domain protein (TRADD) (12, 13), which functions as a scaffold enabling the recruitment of receptorinteracting protein kinase 1 (RIPK1) (14 -16), TNF receptorassociated factor 2 (TRAF2) or TRAF5 (12), and the cellular inhibitor of apoptosis proteins (cIAPs) cIAP1 and cIAP2 (11), which collectively form a signaling composite called complex I (17-19). The resulting lysine 63-linked polyubiquitination of RIPK1 by TRAF2 and the cIAPs (20 -24) enables an interaction with the IB kinase complex that mediates the phosphorylation and degradation of IB-inhibitory proteins and activation of the transcription factor NF-B to promote non-apoptotic signaling pathways (25-27). NF-B also increases expression of anti-apoptotic genes, including cIAPs and FLICE inhibitory protein (c-FLIP), further ensuring a non-apoptotic signaling pathway.The importance of receptor internalization as a regulatory mechanism for the segregation and divergence of intracellular signaling pathways is highlighted by studies on internalization of TNFR1 and TNFR2, the Fas receptor (FasR/CD95), IL-1 receptor I, Toll-like receptor 4, and TRAIL receptors (18, 28 -33). The current favored m...

The importance of presenilin-dependent γ-secretase protease activities in the development, neurogenesis, and immune system is highlighted by the diversity of its substrates and characterization of Psen1- and Psen2-deficient transgenic animals. Functional differences between presenilin 1 (PS1) and presenilin 2 (PS2) are incompletely understood. In this study, we have identified a Psen2-specific function, not shared by Psen1 in Toll-like receptor signaling. We show that immortalized fibroblasts and bone marrow-derived macrophages from Psen2- but not Psen1-deficient mice display reduced responsiveness to lipopolysaccharide (LPS) with decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) activity and diminished pro-inflammatory cytokine production. In whole animal in vivo responses, Psen2-deficient animals have abnormal systemic production of LPS-stimulated pro-inflammatory cytokines. Mechanistically, we demonstrate that Psen2 deficiency is paralleled by reduced transcription of tlr4 mRNA and loss of LPS-induced tlr4 mRNA transcription regulation. These observations illustrate a novel PS2-dependent means of modulating LPS-mediated immune responses and identify a functional distinction between PS1 and PS2 in innate immunity.

Most microbes have developed responses that protect them against stresses relevant to their niches. Some that inhabit reasonably predictable environments have evolved anticipatory responses that protect against impending stresses that are likely to be encountered in their niches–termed “adaptive prediction”. Unlike yeasts such as Saccharomyces cerevisiae, Kluyveromyces lactis and Yarrowia lipolytica and other pathogenic Candida species we examined, the major fungal pathogen of humans, Candida albicans, activates an oxidative stress response following exposure to physiological glucose levels before an oxidative stress is even encountered. Why? Using competition assays with isogenic barcoded strains, we show that “glucose-enhanced oxidative stress resistance” phenotype enhances the fitness of C. albicans during neutrophil attack and during systemic infection in mice. This anticipatory response is dependent on glucose signalling rather than glucose metabolism. Our analysis of C. albicans signalling mutants reveals that the phenotype is not dependent on the sugar receptor repressor pathway, but is modulated by the glucose repression pathway and down-regulated by the cyclic AMP-protein kinase A pathway. Changes in catalase or glutathione levels do not correlate with the phenotype, but resistance to hydrogen peroxide is dependent on glucose-enhanced trehalose accumulation. The data suggest that the evolution of this anticipatory response has involved the recruitment of conserved signalling pathways and downstream cellular responses, and that this phenotype protects C. albicans from innate immune killing, thereby promoting the fitness of C. albicans in host niches.

S9.4 Free oral presentations (late breaking), September 23, 2022, 4:45 PM - 6:15 PM Candida albicans adaptation to host niches affects the exposure of key pathogen-associated molecular patterns (PAMPs) on its cell surface and, consequently, the detection of C. albicans cells by the immune system. Focusing on β-(1,3)-glucan, we screened for host inputs that influence the exposure of this immune-stimulatory PAMP on the C. albicans cell surface. We used a combination of fluorescent microscopy, flow cytometry, and cytokine assays, and then analyzed certain conditions in more detail using transmission electron microscopy and time-lapse video microscopy of C. albicans-phagocyte interactions. We found that some nutrients, micronutrient limitation, stresses, and antifungal drugs trigger β-glucan masking, whereas other inputs, such as nitrogen sources and quorum sensing molecules, exert limited effects on β-glucan exposure. In particular, host- or bacterial-derived L-lactate, hypoxia, or iron limitation induce β-glucan masking, and this leads to attenuation of phagocytic responses [Nature Micro 2, 16 238; mBio 9, e01318-18; Nature Comms 10, 5315]. Lactate signals through Gpr1 to activate Crz1 in a calcineurin-independent manner, whereas hypoxia signals via mitochondrial ROS, and iron limitation signals through Ftr1 and Sef1. β-glucan masking also depends upon downstream signaling via the cAMP-PKA pathway. We conclude that C. albicans has evolved to exploit a range of specific host-derived signals to modulate the exposure of a major PAMP at its cell surface in an attempt to evade phagocytic uptake. Using barcode-sequencing in direct competition assays in vivo, we showed that preadaptation to specific β-glucan masking signals affects the ability of this fungus to colonize particular tissues during systemic infection in a murine model. This reinforces the view that β-glucan masking promotes C. albicans infection.